Monospecific antibodies against a subunit of fibrinogen

ABSTRACT

The invention provides nonspecific antibodies which are specifically reactive with the α E  subunit of fibrinogen or a fragment thereof, but not with other portions of the fibrinogen molecule. The invention also provides anti-α E  probes, including nonspecific anti-α E  antibodies which have been detectably labeled. In addition, the invention provides methods of using the nonspecific antibodies for detection of the α E  subunit and fragments thereof, as well as reagents and kits for performing the methods. Diagnostic methods for determining information associated with atherogenesis and/or thrombogenesis, as well as for determining information associated with pregnancy status or outcome. The invention further provides continuous cell lines which produce monospecific anti-α E  antibodies.

This invention was made in part with Government support under NIH GrantROI HL51050 awarded by the Public Health Service. The Government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

This invention relates to monospecific antibodies which are reactivewith a subclass of fibrinogen. More particularly, the invention relatesto monospecific antibodies which are reactive with the α_(E) subunit ofthis fibrinogen molecule, and to methods of use of such antibodies.

Fibrinogen is one of the more well-studied and abundant proteins in thehuman circulatory system. Its complex structure--a heavilydisulfide-bonded hexamer composed of two copies each of the α, β and γsubunits--and central role in blood clot formation and wound healingaccount for the high profile it has enjoyed as a subject of bothbiochemical and medical research. Recently, new attention has been givento structure/function relationships in the fibrinogen molecule. This newinterest has in part been prompted by growth in the understanding ofthis protein's range of activity in normal and pathological states(Refs. 1-3). However, a major impetus to fibrinogen research has beenprovided by the recent identification of a long overlooked, naturallyoccurring variant of the α subunit, designated "α_(E) " (Ref. 4) Unlikethe α subunit, the α_(E) subunit bears a C-terminal extension whichconfers significant homology to the β and γ subunits.

Fibrinogen is synthesized and secreted into the circulation by theliver. Circulating fibrinogen is polymerized under attack by thrombin toform fibrin, which is the major component of blood clots or thrombi.Subsequently, fibrin is depolymerized under attack by plasmin to restorethe fluidity of the plasma. Many of the steps in the polymerization anddepolymerization processes have been well established (Ref. 5). Theelevated levels of fibrinogen which are part of the acute phase responseoccurring in the wake of infections and trauma are now known to comefrom increased hepatic production, primarily in response tointerleukin-6 (IL-6) (Ref. 6).

By the late 1960's, the general subunit structure of fibrinogen wasfirmly established (Ref. 7) and, a decade later, the complete amino acidsequence was reported (Refs. 8-11). Over the next 10 years, the clusterof three separate genes encoding the α (alpha), β (beta), and γ (gamma)subunits was identified on chromosome 4q23-q32 (Ref. 12), and theapparently complete genetic sequences of all three fibrinogen subunitswere published (Ref. 13). These studies indicated that the α subunitlacked a globular C-terminal domain comparable to those present in the αand γ subunits.

The subsequent discovery of an additional exon (i.e., exon VI)downstream from the established α subunit gene has resolved theevolutionary mystery posed by the imperfectly parallel structure of thethree major subunits (Refs. 4, 14). A novel fibrinogen α chaintranscript has been identified at low frequency bearing the exonVI-derived sequences as a separate open reading frame. Additionalsplicing leads to the use of this extra sequence to elongate the α chainby 35%, providing the subunit with a globular domain (the "VI-domain")similar to those of the β and γ chains. Evidence shows that thispreviously unidentified extended α chain (α_(E)) is assembled intofibrinogen molecules and that its synthesis is enhanced by interleukin-6(IL-6). These facts suggest that the α_(E) subunit participates in boththe acute phase response and in normal physiology.

Using a polyclonal rabbit antibody preparation specific to theVI-domain, α_(E) was demonstrated to occur in plasma fibrinogen as partof (α_(E) βγ)₂, a homodimeric (i.e., symmetrical) molecule of ˜420kilodaltons (kDa) (Ref. 15). This species has been designated "Fib₄₂₀ "to distinguish it from the abundant 340 kDa form of fibrinogen, denoted"Fib₃₄₀ " ((αβγ)₂). Although the relatively low circulating level ofFib₄₂₀ (˜1% of total fibrinogen) is undoubtedly responsible for itshaving escaped detection until recently, the two extra globular domainsare likely to significantly influence the fibrinogen molecule's multiplebinding capacities and functions.

A definitive topology of the fibrinogen molecule awaits resolution ofits elusive crystal structure. However, numerous indirect studies (Ref.5) point to a trinodal structure, in which the amino ends of all of thechains are contained in a central nodule from which two triple chaindisulfide-interlocked coiled coils diverge. These coils lead to twodistal nodules made up of the globular, carboxy terminal domains of theβ and γ subunits. Among the more obvious possible functions of the novelglobular extension of the α chain C-terminus may be protection of the αchain from proteolytic attack. It is also conceivable that such a domainwould alter properties of the protofibrils which constitute thelaterally cross-linked fibrin strands, particularly if the domainprotruded in a plane distinct from that defined by the other threenodules.

Transcripts encoding fibrinogen subunit counterparts havingexceptionally high C-terminal homology to human α_(E) have been detectedthus far in lamprey, where it arises from a second α gene (Refs. 16,17),as well as in chicken, rabbit, rat, and baboon. This degree of αsubunit-associated globular domain preservation in the vertebrate genomesignals an important, if as yet unknown, role for α_(E). Clues to thepotential significance of variations in the α chain may lie in thesimilarity of the extension in α_(E), not only to the correspondingregions of the fibrinogen β and γ chains, but also to carboxy domains atthe C-termini of a number of non-fibrinogen proteins from fruit fly toman (Refs. 18-25). Where functions are known, these non-fibrinogenproteins are constituents of the extracellular matrix and have adhesiveproperties. It is expected that continued research will permit thedetermination of whether the α_(E) globular domain contributes in asubtle way to the primary function of fibrinogen (clot formation andwound healing) or, following the example of other differentially usedexons (Refs 26-28), promotes an alternative function.

In wound repair, fibrinogen serves as a key protein, achieving rapidarrest of bleeding following vessel injury. It promotes both theaggregation of activated platelets with one another to form a hemostaticplug, as well as endothelial cell binding at the site of injury to sealthe margins of the wound. As the most abundant adhesive protein in theblood, fibrinogen attaches specifically to platelets, endothelial cellsand neutrophils via different integrins (Ref. 29). Five putativereceptor recognition domains on human fibrinogen, distributed over itsthree subunits, have been identified by in vitro and in vivo analyses(Refs. 30-35). In fibrinogen which contains the variant α_(E) chains,masking of these sites, as well as addition of new sites, are distinctpossibilities with ramifications that must be explored. Molecular toolsadequate to this purpose have yet to be developed.

Elevated levels of fibrinogen have been found in patients suffering fromclinically overt coronary heart disease, stroke and peripheral vasculardisease. Although the underlying mechanisms remain speculative, recentepidemiological studies leave little doubt that plasma fibrinogen levelsare an independent cardiovascular risk factor possessing predictivepower which is at least as high as that of other accepted risk factorssuch as smoking, hypertension, hyperlipoproteinemia or diabetes (Refs.36, 37). The structure of fibrin has been analyzed extensively in vitro(Ref. 5). Only recently, however, has attention been paid to themolecular structure of human thrombi and atherosclerotic plaques withrespect to fibrinogen and fibrin products (Ref. 38). Whereas thrombiformed in vivo consist primarily of fibrin II cross-linked by factorXIIIa, fibrinogen itself is a major component of uncomplicatedatherosclerotic lesions, particularly fibrous and fatty plaques.Immunohistochemical as well as immunoelectrophoretic analyses indicatethat fibrinogen in the aortic intima is comparatively well protectedfrom thrombin and plasmin, and that much of it is deposited throughdirect cross-linking by tissue transglutaminase without becomingconverted to fibrin (Ref. 39). Further understanding of these issuesawaits the development of methods for the differential determination offibrinogen subtypes in medical samples.

Fibrinogen-derived protein is also a major component of the stroma inwhich tumor cells are embedded, but little is known about its molecularstructure. Tumor cells promote the secretion of potent permeabilityfactors which cause leakage of fibrinogen from blood vessels (Ref. 3).Extravascular clotting occurs due to procoagulants associated with tumorcells. The resulting fibrinogen/fibrin matrix is constantly remodeledduring tumor growth as a consequence of fibrinolysis induced by tumorcell-derived plasminogen activators. It is assumed thatfibrin/fibrinogen degradation products play a role during escape ofmetastatic tumor cells from the primary tumor. There are indicationsthat integrin α_(v) β₃, which is known to interact with the RGDS site inthe C-terminal region of the α chain, may be an important tumor cellsurface receptor since it is preferentially expressed on invasivemelanoma (Ref. 40). It is not known what effect the globular domain ofFib₄₂₀ 's α_(E) subunit plays in tumor development.

Despite evidence indicating roles for α_(E) fibrinogen in a variety ofphysiological processes, it appears that α_(E) deficiency is not lethalin man. This inference is drawn from a recent report on fibrinogenMarburg, a homozygous case of dysfibrinogenemia (Ref. 41). In the α genecoding for this abnormal fibrinogen, a single base substitution (A→T)has been identified that changes codon α 461 AAA (lysine) to TAA (stop).As a result, the carboxy-terminal segment 461 to 625 of the common αchain is lacking and no formation of α_(E) is possible. Symptomsdisplayed by the homozygous propositus consisted of severe hemorrhageafter delivery followed by repeated thrombotic events that occurred,paradoxically, despite unusually low fibrinogen levels. It is not clearwhether the mutant α chain itself, or the lack of α_(E), is responsiblefor these symptoms.

Fibrinogen levels increase during normal pregnancy (Ref. 42). There isclinical evidence that supports the hypothesis that fibrinogen andfirbrin homeostasis is important in pregnancy: low adult levels of(total) fibrinogen were reported to be associated with spontaneousabortions, while fibrinogen infusion was associated with successfulgestation (Refs. 43-45). It is undoubtedly significant that, while thefetal concentration of total fibrinogen at term--as measured inumbilical cord blood plasma--is significantly lower than that of adults,the relative level of the Fib₄₂₀ subclass is dramatically (about 10times) higher. None of these phenomena is understood at the molecularlevel, bespeaking further need for molecular probes with which to definethe role of fibrinogen and its subclasses in the underlyingphysiological mechanisms.

To this time, no stable, sensitive and precise means has existed fordetecting and/or purifying α_(E) -containing fibrinogen (Fib₄₂₀). Asnoted above, a polyclonal antibody has been generated which exhibitsspecificity for the α_(E) subunit (Refs. 4, 15), but such antibodies arenotoriously problematic when employed for analytical and diagnosticapplications. In particular, polyclonal antibodies by their very naturerespond to more than one epitope and, therefore, cannot be used to probeindividual subdomains in structure/function analyses of a molecule.Moreover, the specificity of polyclonal antibodies varies from animal toanimal, as well as with every immunization, as the various antibodysubpopulations fluctuate. Indeed, it is not uncommon that only a singleanimal can be found which is responsive to an immunogen. These problemsprohibit the development of precise, accurate and reproducible methods,tests and diagnostics involving the specific identification of α_(E)subunit of fibrinogen.

As a result, there exists a need for highly specific, sensitive andreproducible probes for enhancing the understanding of the structure andfunction of fibrinogen, especially in relation to the α_(E) subunitthereof. There also exists a need for probes suitable for the detectionand purification of the α_(E) subunit and fibrinogen incorporating thesubunit. In addition, means for diagnostic testing of subjects withrespect to the amount and distribution of fibrinogen in the body areneeded. The present invention effectively addresses these and otherneeds for the first time.

SUMMARY OF THE INVENTION

The present invention provides monospecific antibodies which arereactive with or bind to single epitopes of the α_(E) subunit offibrinogen or a fragment thereof. More particularly, the inventionprovides monospecific antibodies which are reactive with or bind todifferent individual epitopes which occur in the globular domain or theVI-domain of the α_(E) subunit of fibrinogen.

The monospecific antibodies of the invention may include native,modified, or synthetic antibodies. Alternatively, the antibodies mayinclude an antigen-binding region or fragment of a monospecific antibodyspecific for the α_(E) subunit. Thus, the invention includesantigen-binding regions such as Fab, F(ab')₂, and Fv fragments. Theinvention further includes chimeric or hybrid antibodies orantigen-binding regions. Such chimeric compounds may includerecombinant, synthetic and/or natural fragments of the anti-α_(E)monospecific antibodies of the invention which have been combined withother antibody or non-antibody substances.

The invention provides further for monospecific antibodies andantigen-binding fragments thereof, as defined elsewhere herein, whichare attached through methods known in the art to other moieties such asdetectable label moieties and substantially solid substrate materials.For example the invention includes anti-α_(E) antibodies which have beendetectably labeled. Suitable detectable label moieties may be selectedfrom among those known in the art. Substantially solid substratematerials may also be chosen according to the artisan's desired ends.

Further, the invention provides methods for making monospecificantibodies which are specifically reactive with an epitope of the α_(E)subunit of fibrinogen. Principally, such methods include conventionalhybridoma techniques. However, other suitable methods known in the artmay be employed, including approaches such as the use of transgenicanimals in which fibrinogen genes have been altered or supplemented soas to affect the immune response of the animals to fibrinogen. Inaddition, recombinant and molecular biology techniques may be employedin the preparation of hybrid antibodies as desired.

The invention also provides a composition for binding fibrinogen, whichincludes a monospecific antibody or antigen-binding region thereof whichis reactive with or binds an epitope of the α_(E) subunit of fibrinogenor a fragment thereof. The composition may include a pharmacologicallyacceptable carrier and/or other pharmacologically acceptable components,such as carriers, solvents, salts, excipients, physiological substances,bulking agents, and the like. In addition, the composition may includeother components which are separately reactive with fibrinogen, such asother monoclonal or polyclonal antibodies, receptor molecules, orfibrinogen-binding portions thereof. Such compositions may include ananti-α_(E) monospecific antibody of the invention which has beendetectably labeled by a marker moiety. Alternatively, the compositionsmay also include another fibrinogen-binding component, such as ananti-fibrinogen antibody, which has been detectably labeled by the sameor different marker moiety.

The invention further provides a method for binding the α_(E) subunit offibrinogen or a fragment thereof by means of an anti-α_(E) monospecificantibody. Accordingly, Fib₄₂₀ and the α_(E) subunit, natural, modified,and synthetic variants thereof, as well as fragments thereof, may bedetected and measured by means of monospecific antibodies of theinvention.

In the fibrinogen binding method of the invention, the method includescontacting a testable system, in which the presence or absence offibrinogen is to be determined, with a composition having an anti-α_(E)monospecific antibody or antigen-binding region thereof. The method theninvolves measuring an amount of specific association or binding betweenthe testable system and the monospecific antibody. In this method,specific binding of the antibody in the system indicates the presence ofα_(E) subunit-containing fibrinogen in the system. The method of theinvention may be adapted for performance of numerous immunochemicaltechniques.

In a preferred embodiment, the detection method employs a monospecificantibody which has been detectably labeled with a marker moiety. Inother embodiments, the method may employ a monospecific antibody of theinvention which has been bound to a substrate such as a polymericmaterial. In the method, the composition may also include other reagentssuch as other antibodies which differentially detect other fibrinogensubunits or subtypes.

Preferred diagnostic methods according to the invention includedetermining diagnostic information associated with atherogenesis and/orthrombogenesis and determining diagnostic information associated withpregnancy status or outcome. Thus in one preferred embodiment, ininvention includes a method for diagnosing the presence or probabilityof thrombogenesis or atherogenesis in a subject, including:

(a) measuring an amount of fibrinogen in a subject by means of acomposition comprising a monospecific antibody which binds with anepitope of the α_(E) subunit of fibrinogen;

(b) comparing the measured amount of fibrinogen for the subject with anamount of fibrinogen recognized to have an association withthrombogenesis or atherogenesis; and

(c) determining from the comparison in step (b) the presence orprobability of thrombogenesis or atherogenesis in the subject.

In yet another preferred embodiment, the method of the inventionincludes deriving diagnostic information concerning pregnancy status oroutcome, including:

(a) measuring an amount of fibrinogen in a fetal subject by means of acomposition comprising a monospecific antibody which binds with anepitope of the α_(E) subunit of fibrinogen;

(b) comparing the measured amount of fibrinogen for the fetal subjectwith an amount of fibrinogen recognized to have an association with apregnancy status or outcome; and

(c) determining from the comparison of step (b) information concerningpregnancy status or outcome.

The invention further provides diagnostic and experimental kits whichinclude anti-α_(E) monospecific antibody, and enable the detection,purification and/or separation of fibrinogen and the various subtypes orfragments thereof in a specific and reproducible manner. In these kits,the antibodies may be provided with means for binding to detectablemarker moieties or substrate surfaces. Alternatively, the kits mayinclude the antibodies already bound to marker moieties or substrates.

Accordingly, as a result of the invention, there are now providedmonospecific antibodies which are reactive with or bind with epitopes ofthe VI-domain of the α_(E) subunit of fibrinogen or a fragment thereof.There are also provided detectable probes for the detection,localization and purification of fibrinogen and specifically the α_(E)subunit thereof. There are also provided methods for preparingmonoclonal antibodies which are reactive with the VI-domain of the α_(E)subunit of fibrinogen, methods for the preparation of detectableanti-α_(E) monoclonal antibodies, methods for the use of such antibodiesfor the detection, localization and purification of Fib₄₂₀ and relatedcompounds, and methods for the diagnosis and treatment of Fib₄₂₀-related disorders.

These and other advantages of the present invention will be appreciatedfrom the detailed description and examples which are set forth herein.The detailed description and examples enhance the understanding of theinvention, but are not intended to limit the scope of the invention.

BRIEF DESCRIPTION OP THE DRAWINGS

Preferred embodiments of the invention have been chosen for purposes ofillustration and description, but are not intended in any way torestrict the scope of the present invention. The preferred embodimentsof certain aspects of the invention are shown in the accompanyingdrawings, wherein:

FIG. 1 shows a comparative description of the deduced amino acidsequences of the VI-domain of fibrinogen in five species.

FIG. 2 shows a comparative graphical description of the structure of theVI-domain in five species based on hydropathy analysis of amino acidsequences.

FIG. 3 shows a Western blot analysis of human fibrinogen usinganti-α_(E) antibodies.

FIG. 4 shows the aligned amino acid sequences of the human, rodent, andrabbit α_(E) VI-domains

FIG. 5 shows a plot of competition-ELISA using an anti-α_(E) antibody ofthe invention with wild-type and mutant recombinant human VI-domains ascompetitors.

FIGS. 6(a)-6(c) are bar graphs showing binding-ELISA of wild-type,truncated, and mutated recombinant VI-domains (the latter expressed inE. coli) using monospecific anti-α_(E) antibodies of the invention.

FIG. 7 shows competition-ELISA with recombinant VI-domain, and fractionA- and fraction B-fibrinogen as competitors using MoAb #3-10.

FIG. 8 shows a Western blot analysis of adult and fetal fibrinogenlevels by means of a monospecific antibody of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides monospecific antibodies which arereactive with or bind with epitopes of the α_(E) subunit of fibrinogenor a fragment thereof. In particular, the invention providesmonospecific antibodies, such as monoclonal antibodies, which arereactive with the VI-domain of the α_(E) subunit of fibrinogen. Theinvention also provides compositions containing such monospecificantibodies, as well as detectable probes for the detection, localizationand purification of fibrinogen and specifically the α_(E) subunitthereof. Methods for preparing monoclonal antibodies which are reactivewith the VI-domain of the α_(E) subunit of fibrinogen are also provided.Moreover, methods for the preparation of detectable anti-α_(E)monoclonal antibodies, methods for the use of such antibodies for thedetection, localization and purification of Fib₄₂₀ and relatedcompounds, and methods for the diagnosis and treatment of Fib₄₂₀-related disorders, are also provided.

The monospecific antibodies of the invention may exhibit anti-α_(E) oranti-VI reactivity which is independent of the molecular or cellularcontext in which the α_(E) subunit occurs. Therefore, the inventionincludes monospecific antibodies which identify epitopes of the α_(E)subunit or the VI-domain, whether as independent molecules orincorporated into a fibrinogen molecule, whether intracellular orextracellular, or whether naturally occurring (native), modified, orsynthetic (e.g., recombinant). The monospecific antibodies of theinvention may be specifically reactive with a particular form of α_(E),or may be reactive with a native or synthetic fragment thereof.

As used herein, the term "monospecific antibody" refers to anyhomogeneous antibody or antigen-binding region thereof which is reactivewith, preferably specifically reactive with, a single epitope orantigenic determinant. The term "monospecific antibody" most commonlyrefers to a monoclonal antibody, also abbreviated "MoAb", as that termis conventionally understood. The term "monospecific antibody" as usedherein may, however, refer to homogeneous antibodies which are native,modified, or synthetic, and can include hybrid or chimeric antibodies.The term does not include "polyclonal antibodies" as that term iscommonly understood.

The term "antigen-binding region" refers to a naturally occurring,modified, or synthetic fragment of a monospecific antibody of theinvention which is reactive with an epitope of the α_(E) subunit offibrinogen. Such antigen-binding regions include, but are not limitedto, Fab, F(ab')₂, and Fv fragments.

The term "anti-α_(E) " refers to the ability of the monospecificantibodies of the invention to react specifically with α_(E) fibrinogenor the α_(E) subunit thereof. Similarly, the term "anti-VI" refers toantibodies of the invention which are specific to or reactive with theVI-domain of α_(E) fibrinogen.

The term "fibrinogen" without more is intended to include any type offibrinogen. Fibrinogen, therefore, refers to monomeric and dimericfibrinogen molecules having the monomer structure (αβγ), as well asmolecules having the monomer structure (αβγ), and other hybridmolecules, whether naturally occurring, modified, or synthetic. The term"fibrinogen" refers generally to fibrinogen from humans but may includefibrinogen of any species. In addition, the term may be specificallylimited to a particular species in particular contexts, such as "humanfibrinogen".

The term "Fib₃₄₀ " refers to the predominant subclass of humanfibrinogen, which molecules have the homodimeric structure (αβγ)₂, andhave a molecular weight of 340 kilodaltons (kDa) or less. A range ofmolecular weights of fibrinogen with a maximum of about 340 kDa isnormally observed, and is attributed to variations in the lengths of theα subunit tails due to their having been subjected to various amounts ofproteolytic cleavage.

The term "Fib₄₂₀ " refers to the minor subclass of human fibrinogen,which molecules have the homodimeric structure (αβγ)₂, and have amolecular weight of about 420 kDa (Ref. 15). In normal subjects, thistype of fibrinogen occurs with a frequency of about 1% of all fibrinogenin the body. This type of fibrinogen generally does not exhibit muchvariance in molecular weight, probably because the α subunit tail may besubstantially protected from random proteolytic attack by virtue of thepresence of the additional globular domain peculiar to the α_(E)subunit.

It is known in the art that monoclonal antibodies are, in general,difficult to produce. For example, it has been estimated that more than1,000 clones need to be screened to find one or two antibodies which arespecific enough and exhibit enough affinity with the antigen to permituse. These difficulties stem from problems such as irreproducibility ofan initial positive screen, or failure to obtain subclones in the firstcloning. Such problems are commonly related to the deaths of cells,instability in cell lines, low antibody yield in ascites, instability ofantibody, etc.

Despite such common difficulties, there have now been producedmonospecific antibodies against the α_(E) subunit of fibrinogen. Moreparticularly, monoclonal antibodies which are reactive with or bind withthe VI-domain of the α_(E) subunit of fibrinogen have been prepared.This achievement was even more unexpected given the further complicationthat the intended antigen region, i.e., the α_(E) VI-domain offibrinogen, is substantially conserved throughout the animal kingdom. Itis generally known that the development of monoclonal antibodies isunusually difficult to achieve when the targeted antigenic determinantis part of a highly conserved protein or peptide. This is principallybecause various related species tend to possess the same or a closelyrelated protein or peptide, and their immune systems perceive suchproteins or peptides as "self" antigens. In most cases, an animal of onespecies will generate little if any immune response when challenged witha highly conserved protein or peptide which is derived from anotherspecies. As a result, conserved proteins exhibit little or noimmunoreactivity when used to challenge an animal of another species,and the development of antibodies against the protein is correspondinglyminimal.

A measure of inter-species homology for the α_(E) subunit of fibrinogenis illustrated in FIG. 1, in which the amino acid sequence of the humanα_(E) VI-domain is shown in comparison with the corresponding sequencesof four other species (baboon, rabbit, rat, and chicken). The deducedamino acid sequences of the α_(E) VI-domains of man, baboon, rat,rabbit, and chicken align in an uninterrupted block of 236 residues. Thecomplete sequence of amino acids (single letter abbreviations) for thehuman α_(E) VI-domain is shown. The dashes (-) in the sequences of theother species indicate residues which exactly match with thecorresponding residues in the human sequence, while different amino acidresidues are duly indicated by single letter abbreviations.

The human α_(E) VI-domain shares a striking 99%, 94%, 93%, and 76% aminoacid identity, respectively, with its counterparts in baboon, rabbit,rat and chicken. The fact that, in evolutionary terms, at least threequarters of these amino acid positions have remained invariant for 200million years suggests involvement of the VI-domain in a function vitalto the organism.

The VI-domain sequences shown in FIG. 1 were subjected to hydropathyanalysis using the parameters given by Kyte and Doolittle (Ref. 46).That the hydropathy plots of the mammalian VI-domains prove to bevirtually interchangeable, not only with each other but also with theavian domain (FIG. 2), provides a further indication of the domain'sconservation throughout vertebrate evolution.

The skilled artisan will appreciate the extraordinarily high level ofhomology among these sequences. Moreover, the artisan will furtherappreciate, a priori, the likely difficulty-of raising monoclonalantibodies specifically reactive against the human sequence in an animalof another species. Indeed, the difficulty of generating antibodies ofany kind which are free from inter-species cross-reactivity will also beperceived.

In practice, the development of monoclonal antibodies to the VI-domainproved exceptionally difficult. In large part this was due to the highdegree of conservation of the VI-domain and its resulting poorimmunogenicity. Other complications included the unavailability ofpurified Fib₄₂₀, requiring cumbersome screening and characterization ofMoAbs to the VI-domain. these procedures are detailed elsewhere herein.

Nonetheless, despite these difficulties, the present invention provideshybridoma cell lines which produce monoclonal antibodies reactive withepitopes of the α_(E) subunit of fibrinogen and fragments thereof. Theantibodies produced by these hybridomas are also important aspects ofthe invention.

The hybridoma technology originally described by Kohler and Milstein(Ref. 47) can be used to prepare hybridoma cell lines whose secretoryproduct, monoclonal antibodies, are reactive with an epitope orantigenic determinant of the α_(E) subunit of fibrinogen. A generalmethod of preparing these hybridoma cell lines of the invention isdescribed below. Further detail concerning the method is provided in theExamples, which relate the construction of several specific hybridomacell lines. Those skilled in the art will recognize that the presentinvention, including the monoclonal antibodies and hybridoma cell linesdescribed in detail herein, provide a variety of ways to make thehybridomas, and thus the antibodies of the invention. The artisan isreferred to Kennett et al. (Ref. 48) for further details on hybridomatechnology.

Hybridoma cell lines of the invention can be prepared using the α_(E)subunit of fibrinogen or an immunogenic fragment thereof as immunogenicmaterial for activation of immunologically relevant spleen cells. Spleencells are then immortalized by fusion with mouse myeloma cells. Thehybrid cells, called hybridomas, or hybridoma cell lines resulting fromthe fusion, are then selected and screened for reactivity with therecombinant VI-domain.

The anti-α_(E) monospecific antibodies described herein are merelyillustrative of the invention, and all monospecific antibodies which arespecifically reactive with the α_(E) subunit or a fragment thereof,regardless of species of origin or immunoglobulin class or subclassdesignation, including IgG, IgA, IgM, IgE, and IgD, are included in thescope of this invention. The present invention also providesantigen-binding fragments of the anti-α_(E) antibodies. The ability tobind to the α_(E) subunit as opposed to non-α_(E) -derived substances(particularly the predominant α subunit) is a general characteristic ofmonospecific antibodies of the invention.

As discussed above, monospecific antibodies of the invention can beconstructed and isolated by immunization, preparation of hybridomas, andidentification of antibodies with a reactivity to the α_(E) subunit offibrinogen having similarity to that of anti-α_(E) antibodies described.However, the present invention also provides means for identifyingmonospecific antibodies of the invention that does not requiredetermination of antibody reactivity with a broad number α_(E) epitopeor fragments. Antibodies of the invention can be identified also byimmunoprecipitation and competitive binding studies using the antibodyproduced by the cell lines described herein.

Immunoprecipitations using the anti-α_(E) monospecific antibody can beused to determine antigenic identity. Confirmation of identity can beobtained by depleting the antigen from testable samples such as plasmasamples, using excess amounts of one anti-α_(E) antibody and observingthe inability of another antibody to immunoprecipitate an α_(E) fragmentfrom the treated sample. Also, in instances in which the antibodies bindwith the same epitope or closely associated epitopes, each antibody willcompete with the other(s) for binding to the α_(E) subunit. Competitivebinding studies are generally known in the art.

Treatment of antibody preparations with proteolytic enzymes such aspapain and pepsin generates antibody fragments, including the Fab andF(ab')2 species, which retain antigen-binding activity. Treatment of theantibodies of the invention with such enzymes can therefore be used togenerate the α_(E) subunit antigen-binding fragments of the invention.The preparation of antigen-binding fragments of the antibodies of theinvention and their diagnostic and therapeutic usefulness, as well asother applications, suggest themselves to the skilled artisan.Antigen-binding fragments of the anti-α_(E) antibody are especiallyuseful in therapeutic embodiments of the present invention.

Those skilled in the art will recognize that the antigen-binding regionof the antibodies and antibody fragments of the invention is a keyfeature of the present invention. The anti-α_(E) hybridoma cells of theinvention serve as a preferred source of DNA that encodes suchantigen-binding regions of the invention. This DNA, through recombinantDNA technology, can be attached to DNA that encodes any desired aminoacid residue sequence to yield a novel "hybrid", or "chimeric", DNAsequence that encodes a hybrid, or chimeric, protein. In such a fashion,chimeric antibodies of the invention, in which one portion of theantibody is ultimately derived from one species and another portion ofthe antibody is derived from another species, can be obtained. However,the present invention also comprises any chimeric molecule that containsan α_(E) antigen-binding region.

Antibodies of the present invention can also be labeled with anydetectable group, such as fluorescent labels, enzyme labels, andradioactive labels to identify expression of the α_(E) subunit or partsthereof. Detector groups useful according to the invention include, forexample, fluorescein as a fluorescent label, horseradish peroxidase asan enzyme label, and Iodine-125 as a radioactive label. Additionalfluorescent labels which can be utilized in the invention include, butare not limited to, rhodamine, phycoerythrin and additional compoundsemitting fluorescent energy. Additional enzyme labels which can beutilized in this invention include, but are not limited to, glucoseoxidase and alkaline phosphatase. Additional radioactive labels whichcan be utilized in this invention include, but are not limited to,Iodine-131 and Indium-111.

Suitable detectable labels may be selected from among those known in theart, including, but not limited to, radioactive labels, enzymes,specific binding pair components, colloidal dye substances,fluorochromes, reducing substances, latexes, digoxigenin, metals,particulates, dansyl lysine, antibodies, protein A, protein G, electrondense materials, chromophores, and the like. Effectively, any suitablelabel, whether directly or indirectly detectable, may be employed. Oneskilled in the art will clearly recognize that these labels set forthabove are merely illustrative of the different labels that could beutilized in this invention.

Fibrinogen α_(E) subunit-reactive antibodies of the invention can alsobe derivatized by conjugation to biotin, and used, upon addition ofspecies of avidins which have been rendered detectable by conjugation tofluorescent labels, enzyme labels, radioactive labels, electron denselabels, etc., in a multiplicity of immunochemical and immunohistologicalapplications.

The monospecific antibodies of the invention may also be attached orbound to substrate materials according to methods known to those skilledin the art. Such materials are generally substantially solid andrelatively insoluble, imparting stability to physical and chemicaldisruption of the antibodies, and permitting the antibodies to bearranged in specific spatial distributions. Among substrate materials,materials may be chosen according to the artisan's desired ends, andinclude materials such as gels, hydrogels, resins, beads,nitrocellulose, nylon filters, microtiter plates, culture flasks,polymeric materials, and the like, without limitation.

The monospecific antibodies of the present invention, whether labeled orunlabeled, can be used in immunological assays to determine the presenceof α_(E) -containing fibrinogen in tissue samples from human or animalsubjects. Biopsy and necropsy samples of subjects, as well as samplesfrom tissue libraries or blood banks, can be evaluated for the presenceof α_(E) -containing fibrinogen using an anti-α_(E) antibody of thisinvention. Moreover, suitable pharmaceutical preparations according tothe invention may be employed for in vivo use, such as for thevisualization of fibrinogen or fibrinogen-containing substances andstructures in a living subject.

Thus, the invention provides a method for binding the α_(E) subunit offibrinogen or a fragment thereof by means of an anti-α_(E) monospecificantibody. Accordingly, Fib₄₂₀ and the α_(E) subunit, natural, modified,and synthetic variants thereof, as well as fragments thereof, may bedetected and measured by means of monospecific antibodies of theinvention.

In the fibrinogen binding method of the invention, the method includescontacting a testable system, in which the presence or absence offibrinogen is to be determined, with a composition comprising ananti-α_(E) monospecific antibody or antigen-binding region thereof. Themethod then involves measuring an amount of specific association orbinding between the testable system and the monospecific antibody. Inthis method, specific binding of the antibody in the system indicatesthe presence of α_(E) subunit-containing fibrinogen in the system. Thetestable system may be either in vivo or in vitro, and the method of theinvention may be performed in vivo, in vitro, or a combination thereof.

In a preferred embodiment, the detection method employs a monospecificantibody which has been detectably labeled with a marker moiety. Inother embodiments, the method may employ a monospecific antibody of theinvention which has been bound to a substrate material. In the method,the composition may also include other reagents such as other antibodieswhich differentially detect other fibrinogen subunits or subtypes.

The fibrinogen binding method of the invention includes methods known inthe art which employ antibodies to specifically bind target substances.Preferred methods include immunochemical methods, such as enzyme-linkedimmunosorbent assay (ELISA) methods, immunonephelometry methods,agglutination methods, precipitation methods, immunodiffusion methods,immunoelectrophoresis methods, immunofluorescence methods, andradioimmunoassay methods.

The invention further includes a method for determining or diagnosingthe existence of or probability of thrombogenesis or atherogenesis in asubject. Alternatively, the method includes the detection andlocalization of fibrotic or atherosclerotic plaques and/or lesions. Inthis method, an amount of fibrinogen is measured by means of acomposition including an anti-α_(E) monospecific antibody of theinvention. The measured amount of fibrinogen is compared with an amountof fibrinogen which is recognized or known to be associated withthrombogenesis or atherogenesis. The method then involves thedetermination from the measured and standard value(s) of the presence orlikelihood of thrombogenesis or atherogenesis in the subject. The methodcan include measuring or detecting fibrinogen in vivo, such as byimaging or visualizing the location and/or distribution of fibrinogen,and especially α_(E) fibrinogen, in the body. Alternatively, the methodincludes obtaining a medical sample from the subject and measuringfibrinogen ex vivo or in vitro. This method preferably involves thedifferential measurement of at least two subtypes of fibrinogen,including α_(E) -containing fibrinogen.

The invention also includes a method for fractionation of fibrinogen.Such methods include contacting a medical sample containing fibrinogenwith a composition of the invention which includes an anti-α_(E)monospecific antibody. Preferably, the method is performed usingconditions which are conducive to binding of fibrinogen with themonospecific antibody. Then the bound fibrinogen is removed from thesample. The method is represented by chromatography-type methods, bothpreparative and analytical. Numerous such methods are known in the artand can be selected by the artisan as desired. In this method, themonospecific antibody may be soluble, suspended in fluid phase, orattached to a substantially solid phase, as desired.

The invention further provides diagnostic and experimental kits whichinclude anti-α_(E) monospecific antibody, and enable the detection,purification and/or separation of fibrinogen and the various subtypes orfragments thereof in a specific and reproducible manner. In these kits,the antibodies may be provided with means for binding to detectablemarker moieties or substrate surfaces. Alternatively, the kits mayinclude the antibodies already bound to marker moieties or substrates.The kits may further include positive and/or negative control reagentsas well as other reagents for adapting the use of the antibodies of theinvention to particular experimental and/or diagnostic techniques asdesired. The kits may be prepared for in vivo or in vitro use, and maybe particularly adapted for performance of any of the methods of theinvention.

The following examples are intended to assist in a further understandingof the invention. The particular materials and conditions employed areintended to be further illustrative of the invention and are notlimiting upon the reasonable scope thereof.

EXAMPLE 1

Production and characterization of anti-VI MoAbs

Monospecific antibodies (MoAbs) specific for the VI-domain of α_(E) weresought for their potential use in column-purification of Fib₄₂₀, inimmunohistochemical analysis, and in developing an ELISA for measuringthe protein in blood samples. Epitope mapping will add these probes tothe formidable arsenal of anti-fibrinogen MoAbs, currently availablefrom several sources, to facilitate dissection of Fib₄₂₀ 'sstructure-function relationships.

Recombinant VI-domain was expressed in E. coli, according to techniquesknown in the art (Ref. 15). Mice were given subcutaneous injection ofeither the partial or complete VI-domain amino acid sequence. Thepartial sequence included 213 amino acids (Gly635-Gln847), and the fullsequence included 236 amino acids (Asp612-Gln847). Hybridoma fusionswere performed according to standard procedures.

Supernatants of parental clones were initially screened by binding-ELISA(B-ELISA) with recombinant VI-domain. Positive clone supernatants werethen further examined for α_(E) -specificity by B-ELISA withfractionated fibrinogen. Potential α_(E) -specificity was then confirmedby establishing that exclusively the Fib₄₂₀ -subclass wasimmunoprecipitated from metabolically labeled HepG2 culture medium byevaluating the subunit on SDS-PAGE. Western analysis of reduced plasmafibrinogen was then performed to establish that the MoAb did recognizeonly the α_(E) -subunit of Fib₄₂₀. Only cell populations withsupernatants recognizing α_(E) in native and denatured Fib₄₂₀ by thesecriteria were subcloned and their MoAbs subtyped. Subclones wereamplified as ascites, and the MoAbs were purified by known techniques(E-Z-SEPTM™ antibody purification kit from Pharmacia Biotech(Piscataway, N.J.), as well as size exclusion chromatography and HPLC)from the ascites fluids. The purified MoAbs were then labeled withhorseradish peroxidase (HRPO) by known methods. As a result of thisscreening procedure, three MoAbs were eventually identified whichexhibited desirable anti-α_(E) specificity. These MoAbs were obtainedfrom hybridomas designated #3-10, #29-1, and #148-B.

EXAMPLE 2

A Western analysis using HRPO-labeled MoAb #3-10 is shown in FIG. 3, toillustrate the specificity of the anti-VI MoAbs of the invention. Inthis analysis, fibrinogen was isolated from normal (adult) plasma, andfrom umbilical cord (fetal) plasma. Adult (100 μg) and fetal (80 μg)fibrinogen, as well as recombinant VI-domain (25 μg), were separated byPAGE under reducing conditions. In FIG. 3, the adult fibrinogen is shownin Lanes 1 and 4, fetal fibrinogen is shown in Lanes 2 and 4, andrecombinant VI-domain is shown in Lanes 3 and 6. Lanes 1-3 are stainedfor protein, while lanes 4-6 are blotted with MoAb #3-10. It is clearthat only α_(E) is recognized from among all the fibrinogen chainspresent. These results clearly demonstrate that the invention providesdistinct new tools for the immunological detection and analysis of theα_(E) subunit of fibrinogen.

EXAMPLE 3

To aid in epitope mapping as well as to provide an alternative approachto generating anti-VI MoAbs, we selected several linear sequences of thehuman VI-domain based both on their immunogenic potential in rodents andrabbits (i.e., significantly different amino acid composition from thealigned sections of these species' α_(E) C-terminal domains) and ontheir cell surface location as predicted by hydropathy analysis. Twolinear peptides were synthesized according to known techniques. Thesepeptides were designated Region 1 (Phe677-Gly691) and Region 2(Gly821-Gln847). The regions are indicated in the shaded portions ofFIG. 4 which displays the amino acid sequences of the human, rodent, andrabbit α_(E) VI-domains. The amino acid sequences were deduced from thehuman exon VI nucleotide sequences (Ref. 4), and from homologous exon VIsequences of rat and rabbit. Residues identical to the human areindicated by a dash (-). Numbers are provided for the human α_(E)sequence. The complete rabbit sequence was not determined.

EXAMPLE 4

Use of the synthetic peptides prepared in Example 3 in dot blot analysisand ELISA implicated two epitopes: one near the VI-domain's N-terminus,which is recognized by MoAb #3-10 (Region 1), and one closer to theC-terminus, recognized by MoAbs #29-1 and #148-B (Region 2).

The specificity of these assays, which require notoriously high molarconcentrations of the small synthetic peptides, was verified using pointmutations and truncations of the VI-domain that were generated andexpressed in E. coli according to methods known in the art. FIG. 5 showsthat mutation of Region 1 of the α_(E) -VI-domain (i.e., 683 E→K, inimitation of the rodent sequence) renders the mutant ineffective as acompetitor in C-ELISA with MoAb #3-10. With MoAb #29-1, by contrast,wild-type and mutant domains were found to be equally potent (data notshown). The results confirm Region 1 of the VI-domain as including theepitope recognized by #3-10.

EXAMPLE 5

Binding-ELISA (B-ELISA) assays using truncations of the VI-domain (FIGS.6(a)-(c)) corroborated and extended the findings shown in Example 4 tosuggest the sites recognized by the other two MoAbs. Microtiter wellswere coated with different recombinant VI-domains as indicated on theabscissa of each panel. "S" indicates the "short", partial VI-domain(residues 635-847); "L" indicates the "long", complete VI-domain(residues 612-847); "S*" indicates the S partial VI-domain which hadbeen mutated in Region 1 (683 E→K); the prefix "N" indicates theN-terminal half of the domain (terminating with residue 717); and "C"represents their common C-terminal halves (beginning with residue 717).

FIG. 6(a) shows that MoAb #3-10 bound significantly only to N-terminalsegments that include an intact region 1 (NS and NL), but not to aC-terminal segment (C) nor to the region 1-mutant (S*). Conversely,MoAbs #29-1 (FIG. 6(b)) and #148-B (FIG. 6(c)) failed to bind theN-terminal segments (NS and NL), but do bind both the C-terminal segment(C) and the mutant (S*). The latter observation supports the notion thatthe epitope for MoAbs #29-1 and #148-B lies in the C-terminal region.All of the MoAbs identified the short (S) and long (L) domains, asexpected.

EXAMPLE 6

To separate Fib₄₂₀ from Fib₃₄₀, a one-step procedure using ion-exchangechromatography has yielded two fibrinogen fractions. Commerciallyavailable fibrinogen preparations (available from, e.g., Sigma ChemicalCo., St. Louis, Mo., and American Diagnostica, Greenwich, Conn.) werefractionated by DEAE-cellulose chromatography. Western blots were usedto characterize each fraction with regard to the presence of Fib₄₂₀. Onefraction, Fraction A, was found to contain no Fib₄₂₀. In the otherfraction, Fraction B, Fib₄₂₀ was found to constitute 10-15% of theprotein (about 50% of this fraction is fibrinogen).

The competitive strength of Fractions A and B in C-ELISA was compared tothat of the recombinant VI-domain. FIG. 7 is a plot illustrating theC-ELISA data. It is shown that Fraction A was not reactive in thissystem, whereas Fraction B shows typical concentration-dependentcompetition. Recombinant VI-domain was approximately 100-fold morereactive than Fraction A. The C-ELISA data are, therefore, consistentwith the Western blot observations for Fractions A and B.

EXAMPLE 7

Identification of the glycosylation site of α_(E)

In contrast to the predominant human α chain which has no carbohydrate,human α_(E) is N-glycosylated (Ref. 15). Of the two potentialglycosylation sites in its VI-domain, the tripeptide at Asn667 isconserved in all vertebrates (including lamprey), while the second, atAsn812, is conserved only in mammals (FIG. 1). By mutation of thesesites (Asn→Gln) using conventional techniques, we found that COS cellsattach carbohydrate only to Asn667 and that this conserved site is alsocritical for proper Fib₄₂₀ assembly and secretion.

EXAMPLE 8

Recombinant α_(E) -fibrinogen and its subdomains

Recombinant Fib₄₂₀ as well as its subdomains may be generated by methodsknown in the art, preferably in yeast such as Saccharomyces cerevisiae,and Pichia pastoris. Using the Pichia system, our laboratory hassucceeded in expressing soluble VI-domains which appear to beglycosylated. It should be noted that solubilization of thenon-glycosylated bacterial recombinant VI-domain was achieved only afterdenaturation with urea. It is believed that the soluble recombinantdomain from yeast folds similarly to the domain in native Fib₄₂₀. Thismaterial can serve as an antigen for developing a new generation ofMoAbs that are capable of recognizing alternative (also non-linear)epitopes and/or are better suited to immunopurification of native Fib₄₂₀from plasma. The soluble wild-type domain and its mutants are alsoextremely important as probes with which to examine the participation ofFib₄₂₀ in polymerization and cross-linking and to define the molecule'ssuspected role as ligand of integrins and other receptors involved incell-cell and cell-matrix interactions.

EXAMPLE 9

Human and fetal fibrinogen was analyzed by Western blot, using the MoAb3-10 according to the invention. Plasma samples, prepared according tostandard procedures from CPDA-1 adult blood and umbilical cord blood,were subjected to electrophoresis under reducing conditions. Fourrepresentative samples of adult and fetal plasma are shown in FIG. 8,with the amount of total fibrinogen being approximately equal in eachlane. Fib₄₂₀ and total fibrinogen levels were determined by measuringα_(E) and β subunits by Western analysis using anti-VI (MoAb 3-10) oranti-β (MoAb Ea3 (Ref. 49)), respectively. The left panel of FIG. 8illustrates Fib₄₂₀ (1 μL adult plasma; 3 μL fetal plasma), and the rightpanel illustrates total fibrinogen (10 nL adult plasma; 30 nL fetalplasma). FIG. 7 shows that Fib₄₂₀ is present as a much larger(approximately 10 times) proportion of fibrinogen in fetal plasma thanin adult plasma.

Thus, while there have been described what are presently believed to bethe preferred embodiments of the present invention, those skilled in theart will realize that other and further embodiments can be made withoutdeparting from the spirit of the invention, and it is intended toinclude all such further modifications and changes as come within thetrue scope of the claims set forth herein.

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What is claimed is:
 1. A monospecific antibody, which binds with anepitope of the α_(E) subunit of fibrinogen, wherein said monospecificantibody is produced by a hydridoma cell line selected from the groupconsisting of hybridoma cell line identified as #3-10, hybridoma cellline identified as #29-1, and hybridoma cell line #148-B.
 2. Themonospecific antibody of claim 1, wherein said epitope occurs in theVI-domain of the α_(E) subunit of fibrinogen.
 3. The monospecificantibody of claim 1, wherein said epitope occurs in the globular domainof the α_(E) subunit of fibrinogen.
 4. The monospecific antibody ofclaim 1, wherein said monospecific antibody is labeled with a detectablemoiety.
 5. The monospecific antibody of claim 4, wherein said detectablemoiety is selected from the group consisting of radioactive labels,enzymes, specific binding pair components, colloidal dye substances,fluorochromes, reducing substances, latexes, digoxigenin, metals,particulates, dansyl lysine, antibodies, protein A, protein G, electrondense materials, and chromophores.
 6. The monospecific antibody of claim1, wherein said monospecific antibody is attached to a substrate.
 7. Themonospecific antibody of claim 6, wherein said substrate includes acomponent selected from the group consisting of gels, hydrogels, resins,beads, nitrocellulose, nylon filters, microtiter plates, culture flasks,and polymeric materials.
 8. The monospecific antibody of claim 1,wherein said monospecific antibody comprises an antigen binding region.9. The monospecific antibody of claim 8, wherein said antigen bindingregion comprises a region selected from the group consisting of Fab,F(ab')₂, and Fv fragments.
 10. The monospecific antibody of claim 1,wherein said antibody is a modified antibody.
 11. The monospecificantibody of claim 1, wherein said antibody is a monoclonal antibody. 12.A composition for binding fibrinogen, comprising a monospecific antibodythereof which binds with an epitope of the α_(E) subunit of fibrinogenwherein said monospecific antibody is produced by a hydridoma cell lineselected from the group consisting of hybridoma cell line identified as#3-10, hybridoma cell line identified as #29-1, and hybridoma cell line#148-B.
 13. The composition of claim 12, wherein said epitope occurs inthe VI-domain of the α_(E) subunit of fibrinogen.
 14. The composition ofclaim 12, wherein said epitope occurs in the globular domain of theα_(E) subunit of fibrinogen.
 15. The composition of claim 12, whereinsaid monospecific antibody is labeled with a detectable moiety.
 16. Thecomposition of claim 15, wherein said detectable moiety is selected fromthe group consisting of radioactive labels, enzymes, specific bindingpair components, colloidal dye substances, fluorochromes, reducingsubstances, latexes, digoxigenin, metals, particulates, dansyl lysine,antibodies, protein A, protein G, electron dense materials, andchromophores.
 17. The composition of claim 12, wherein said monospecificantibody is attached to a substrate.
 18. The composition of claim 17,wherein said substrate includes a component selected from the groupconsisting of gels, hydrogels, resins, beads, nitrocellulose, nylonfilters, microtiter plates, culture flasks, and polymeric materials. 19.The composition of claim 12, wherein said monospecific antibodycomprises an antigen binding region.
 20. The composition of claim 19,wherein said antigen binding region of a monospecific antibody comprisesa region selected from the group consisting of Fab, F(ab')₂, and Fvfragments.
 21. The composition of claim 12, wherein said monospecificantibody is a modified antibody.
 22. The composition of claim 12,wherein said antibody is a monoclonal antibody.
 23. The composition ofclaim 12, wherein said composition further comprises a differentiatingcomponent which binds with Fib₃₄₀.
 24. The composition of claim 23,wherein said differentiating component comprises an anti-Fib₃₄₀ antibodywhich binds with Fib₃₄₀.
 25. The composition of claim 22, wherein saidanti-Fib₃₄₀ antibody is labeled with a detectable moiety.
 26. Thecomposition of claim 24, wherein said anti-Fib₃₄₀ antibody is attachedto a substrate.
 27. The composition of claim 12, further comprising apharmaceutically acceptable substance selected from the group consistingof carriers, solvents, salts, excipients, physiological substances, adbulking agents.
 28. A method of detecting fibrinogen, said methodcomprising:contacting a testable system with a composition comprising amonospecific antibody which binds with an epitope of the α_(E) subunitof fibrinogen wherein said monospecific antibody is produced by ahydridoma cell line selected from the group consisting of hybridoma cellline identified as #3-10, hybridoma cell line identified as #29-1, andhybridoma cell line #148-B, and measuring specific binding of saidmonospecific antibody in said testable system; wherein specific bindingof the monospecific antibody in said testable system is associated withthe presence of fibrinogen in said sample.
 29. The method according toclaim 28, wherein said said testable system is an in vitro testablesystem.
 30. The method of claim 28, wherein said method is animmunochemical method.
 31. The method of claim 30, wherein saidimmunochemical method is selected from the group consisting ofenzyme-linked immunosorbent assay methods, immunonephelometry methods,agglutination methods, precipitation methods, immunodiffusion methods,immunoelectrophoresis methods, immunofluorescence methods, andradioimmunoasay methods.
 32. The method of claim 28, wherein saidepitope occurs in the VI-domain of the α_(E) subunit of fibrinogen. 33.The method of claim 28, wherein said epitope occurs in the globulardomain of the α_(E) subunit of fibrinogen.
 34. The method of claim 28,wherein said monospecific antibody is labeled with a detectable moiety.35. The method of claim 34, wherein said detectable moiety is selectedfrom the group consisting of radioactive labels, enzymes, specificbinding pair components, colloidal dye substances, fluorochromes,reducing substances, latexes, digoxigenin, metals, particulates, dansyllysine, antibodies, protein A, protein G, electron dense materials, andchromophores.
 36. The method of claim 28, wherein said monospecificantibody is attached to a substrate.
 37. The method of claim 36, whereinsaid substrate includes a component selected from the group consistingof gels, hydrogels, resins, beads, nitrocellulose, nylon filters,microtiter plates, culture flasks, and polymeric materials.
 38. Themethod of claim 28, wherein said monospecific antibody comprises anantigen binding region of a monospecific antibody.
 39. The method ofclaim 38, wherein said antigen binding region comprises a regionselected from the group consisting of Fab, F(ab')₂, and Fv fragments.40. The method of claim 28, wherein said antibody is a modifiedantibody.
 41. The method of claim 28, wherein said antibody is amonoclonal antibody.
 42. A kit for the detection of fibrinogen,comprising:(a) a composition comprising a monospecific antibody whichbinds with an epitope of the α_(E) subunit of fibrinogen wherein saidmonospecific antibody is produced by a hydridoma cell line selected fromthe group consisting of hybridoma cell line identified as #3-10,hybridoma cell line identified as #29-1, and hybridoma cell line #148-B;and (b) a container housing said composition.
 43. The kit of claim 42,wherein said monospecific antibody is labeled with a detectable moiety.44. The kit of claim 42, wherein said nonspecific antibody is attachedto a substrate.
 45. The kit of claim 42, wherein said monospecificantibody comprises an antigen binding region of a monospecific antibody.46. The kit of claim 45, wherein said antigen binding region comprises aregion selected from the group consisting of Fab, F(ab')₂, and Fvfragments.
 47. The kit of claim 42, wherein said antibody is a modifiedantibody.
 48. The kit of claim 42, wherein said antibody is a monoclonalantibody.
 49. A method for diagnosing the presence or probability ofthrombogenesis or atherogenesis in a subject, comprising:(a) measuringan amount of fibrinogen in a subject by means of a compositioncomprising a monospecific antibody which binds with an epitope of theα_(E) subunit of fibrinogen wherein said monospecific antibody isproduced by a hydridoma cell line selected from the group consisting ofhybridoma cell line identified as #3-10, hybridoma cell line identifiedas #29-1 and hybridoma cell line #148-B; (b) comparing the measuredamount of fibrinogen for said subject with an amount of fibrinogenrecognized to have an association with thrombogenesis or atherogenesis;and (c) determining from said comparison the presence or probability ofthrombogenesis or atherogenesis in said subject.
 50. A method forderiving diagnostic information concerning pregnancy status or outcome,comprising:(a) measuring an amount of fibrinogen in a fetal subject ofsaid pregnancy by, means of a composition comprising a monospecificantibody which binds with an epitope of the α_(E) subunit of fibrinogenwherein said monospecific antibody is produced by a hydridoma cell lineselected from the group consisting of hybridoma cell line identified a#3-10, hybridoma cell line identified as #29-1, and hybridoma cell line#148-B; (b) comparing the measured amount of fibrinogen for said fetalsubject with an amount of fibrinogen recognized to have an associationwith a pregnancy status or outcome; and (c) determining from saidcomparison information concerning pregnancy status or outcome.
 51. Acontinuous cell line which produces a monospecific antibody which bindswith an epitope of the α_(E) subunit of fibrinogen wherein saidmonospecific antibody is produced by a hydridoma cell line selected fromthe group consisting of hybridoma cell line identified as #3-10,hybridoma cell line identified as: 29-1 and hybridoma cell line #148-B.